Transfer of cargo between regions of the Golgi has properties of diffusion and flow A. Cargo recovers into photobleached Golgi elements. Transiently expressed VSVG3-SP-YFP cargo was accumulated in the ER and pulsed into the secretory pathway of PtK2 cells as in Figure 1B. After 55 minutes at 32°C, (well past the period when cargo is entering the Golgi from the ER), a region covering several Golgi elements was bleached in the YFP channel. The CFP channel was monitored but not bleached. After the bleach, both channels were imaged at 6 second intervals for 15 additional minutes. The total intensity of cargo within all Golgi elements decreases because cargo continues to exit the Golgi during this time, but the relative level of cargo in the bleached region with respect to unbleached region increases, indicating exchange of cargo between the bleached and unbleached regions. Figure 7A corresponds to frames from the movie in Additional Fileset 7. Bar: 10 μm. B. Cargo exchange between Golgi regions is consistent with two underlying processes. Quantitation of the experiment in A as described in Materials and Methods. Cargo fluorescence is normalized for overall loss of cargo from the Golgi due to Golgi exit and for fluctuations due to cell movement and recovery is plotted against time (open circles). The equation shown was fit to the data (line). Due to normalization, the average pre-bleach value of cargo and normalized Golgi resident fluorescence is 1. The analysis relies on the assumption that cargo exits the Golgi equivalently from the bleached an unbleached region, and that CFP and YFP fluorescence remains constant during the experiment (Figure 1). The data are fit well by the equation shown, indicating recovery can be described by sum of linear and exponentially decaying processes.